Pathogenic organisms must accomplish several tasks in order to cause disease, including the secretion of virulence factors. Gram-negative bacteria, however, have two membranes and a space in between that creates a formidable obstacle to secretion. The autotransporters (ATs), a family of proteins characterized by a signal sequence at the N-terminus that directs them to the periplasmic space, a C-terminal domain that forms a barrel in the outer membrane, and a passenger domain that is translocated through this barrel, provide a remarkably simple solution for this problem. However, outer membrane translocation by ATs is not well understood. The autotransporters can be further divided into subfamilies. One of these is the Serine Protease Autotransporters of Enterobacteriaceae (SPATES). These are secreted virulence factors that possess a serine protease motif in their passenger domains. This proposal seeks to study the mechanism of secretion of EspP, a member of this subfamily, as a model for the SPATES. Our hypothesis is that EspP will have a linker region that aids in efficient translocation of the passenger domain through the outer membrane. The first aim seeks to define the predicted secondary structure of this linker. The second aim is to study the relationship between linker structure and function both for efficient outer membrane translocation and insertion of the C-terminus into the outer membrane. These studies will help in accomplishing the long-term goals of understanding autotransporter secretion so that its potential use for antigen presentation in live vaccines can be developed. They will also provide a clearer understanding of gram-negative pathogenesis.